Methods and materials for capture antibody targeted fluorescent in-situ hybridization (cat-fish)

ABSTRACT

The subject invention concerns materials and methods for detecting a target cell in a population. Methods of the invention comprise internally labeling cells via fluorescence in situ hybridization (FISH) using probes that target rRNA, followed by binding of capture antibodies targeted (CAT) for specific cell surface epitopes on the target cells. In one embodiment, the target cells are bacterial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/336,800, filed Dec. 23, 2011, which claims the benefit of U.S.Provisional Application Ser. No. 61/465,227, filed Mar. 16, 2011, and61/428,468, filed Dec. 30, 2010, each of which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, or drawings.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberW911SR-07-C-0084 awarded by the U.S. Army. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Pathogen detection using biosensors is commonly limited due to the needfor both sensitivity and specificity in detecting targets within themixed populations present in complex samples (Lim et al., 2005). Therelatively high detection limits inherent in most systems are influencedby many factors, including low target concentrations, poor captureefficiencies, non-target detection (false positives/negatives) andinterference by organic/inorganic constituents, all of which precludesadequate detection (Simpson-Stroot et al., 2008). Additionally, forthose systems using immunochemistry for target capture or reporting(e.g., antibody sandwich assays), non-target cross-reactivity issues canbe problematic, and adequately specific antibodies are often notavailable.

Some of these limitations may be overcome by combining two differentmolecular signature techniques, which would bestow added confidence foridentifying the presence of targeted pathogens—in particular, the use ofspecifically-targeted fluorescently-labeled 16S rRNA geneoligonucleotide probes in conjunction with specifically-targetedantibodies. The dual level specificity (nucleic acid and protein) allowstwo levels of accuracy for detection and/or confirmation, as well asaddressing cross-reactivity. For example, if the antibody available fora given target cross-reacts with other bacteria (related or otherwise),it could still be used for antibody capture-dependent biosensors, aslong as a labeled nucleic acid specific probe was used to generate thesignal (as opposed to a labeled detector antibody). This probe wouldonly provide fluorescent signal to the appropriate target. Thus, thebinding of unlabeled non-target cells to the antibodies becomes a nullissue as no signal is generated.

The use of fluorescence in situ hybridization (FISH) to phylogeneticallyidentify microorganisms without cultivation based on either 16S or 23SrRNA has become a mainstay of microbial ecology since its introduction(DeLong et al., 1989; Amann et al., 1990; Amann, 1995; Amann et al.,2001; Wagner et al., 2003; Daims et al., 2005). FISH has also beenreported as a rapid method for pathogen identification in clinical andfood settings (Kempf et al., 2000; Hartmann et al., 2005; Kempf et al.,2005; Peters et al., 2006; Wellinghausen et al., 2006; Bisha andBrehm-Stecher, 2009; Bisha and Brehm-Stecher, 2009). Although thepredominant FISH application has been to study microbial communitystructure and spatial arrangements on solid supports, some applicationshave explored its usefulness for flow cytometric analyses (Amann et al.,1990; Wallner et al., 1993; Wallner et al., 1997; Fuchs et al., 1998;Hartmann et al., 2005; Kempf et al., 2005). This adaptation to a liquidphase processing for flow cytometry lends itself to facilitatingbiosensor applications, provided that conditions allowing for both probeand antibody recognition are met.

Traditionally, samples to be processed by FISH are fixed withparaformaldehyde (PFA) to stabilize and preserve them (Daims et al.,2005). PFA acts as a strengthening agent on the membranes ofGram-negative bacteria by cross-linking proteins to prevent lysis duringhybridization, but can make Gram-positive bacteria highly resistant toprobe uptake (Leong, 1994; Daims et al., 2005). Additionally, thiscross-linking activity, while giving stability and excellent conditionsfor FISH, can severely inhibit any subsequent immunochemistry. Tocircumvent these problems, combined bacterial applications of FISH andimmunostaining have typically involved extensive antibody incubationtimes or involved processing steps to overcome the fixative effects(Aβmus et al., 1997; Li et al., 1997; Ramage et al., 1998; Oerther etal., 1999), limiting their utility for rapid and high-throughput testingsituations.

As formaldehyde and its derivatives are well known in the histopathologycommunity to inhibit molecular analyses (e.g., immunostaining,immunohistochemistry or nucleic acid analysis), alternative tissuefixatives have been explored that are more conducive to downstreamprocessing (Baumgärtner et al., 1988; Leong, 1994; Shibutani et al.,2000; Srinivasan et al., 2002; Cox et al., 2006). Methacarn solution hasbeen found to be a non-cross-linking protein-precipitating fixative thatdoes not appear to affect polynucleotide or protein analysis of fixedtissues and usually will give superior immunohistochemical results(Shibutani and Uneyama, 2002). This success with tissues suggests thatmethacarn solution may also be successful with fixation of bacterialcells and lend itself to facilitating the use of FISH in combinationwith immunolabeling for biosensor detection.

In the work described herein, we demonstrate a modified liquid FISHprocessing method used in conjunction with capture antibody targeteddetection (CAT-FISH) to increase the specificity for biosensor assays.Detection of pathogens in pure cultures and seeded matrices wasdemonstrated on a cytometric bead array biosensor, using bead-boundcapture antibodies with FISH labeled cells. Since the applications ofboth FISH and immunochemistry have been well established for use withcomplex sample matrices, this method should be easily adapted to otherbacteria and biosensor platforms. The use of FISH in conjunction withantibody based biosensor assays for pathogen detection has not beenpreviously reported.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for detecting atarget cell in a population. Methods of the invention compriseinternally labeling cells via FISH using probes that target rRNA, suchas 16S and/or 23S rRNA of prokaryotes, followed by binding of captureantibodies targeted (CAT) for specific cell surface epitopes. In oneembodiment, the target cells are bacterial cells. In one embodiment,superparamagnetic beads and IMS-based separations were used as theplatform for target capture in the present invention.

In the present invention, xMAP technology/cytometric bead array wasmodified and used for detection of the FISH labeled cells. In oneembodiment, orange fluorescent superparamagnetic microspheres areconjugated to a specific antibody, the antibody-bead complex is used todraw FISH-labeled target cells out of solution, and the FISH-label isthen read, e.g., by the specialized flow cytometer. The combination ofCAT-FISH and xMAP technology allows elimination of the need for thereporter antibody step, thus simplifying the detection process, reducingnon-specific background signal (improving signal to noise ratios) andreducing costs.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

FIGS. 1A-1F. Comparison of relative fluorescence intensities for E. coliO157 and S. aureus treated with different fixatives. All cells werelabeled with EUB338 Cy3 rRNA FISH probe. Cells fixed with either PFA ormethacarn were fixed prior to mounting on slides for FISH processing.Cells treated via CAT-FISH were mounted on slide after the process wascomplete. FIGS. 1A and 1D) PFA treatment; FIGS. 1B and 1E) Methacarntreatment; FIGS. 1C and 1F) CAT-FISH treatment. Scale bar is 5 μm.

FIGS. 2A-2D. ELISA comparison of fixative effect on antibody binding forliquid FISH hybridized bacteria. FIG. 2A) Pre-hybridized E. coli O157:H7fixed with methacarn or PFA and unfixed control; FIG. 2B)Post-hybridized fixed E. coli O157:H7 after liquid FISH treatment steps;FIG. 2C) Pre-hybridized S. aureus fixed with methacarn or PFA andunfixed control; FIG. 2D) Post-hybridized fixed S. aureus after liquidFISH treatment steps.

FIGS. 3A-3F. CAT-FISH dual labeled E. coli O157:H7 and S. aureus. FIG.3A) E. coli labeled internally with EUB338 CY3 16S rRNA FISH probe; FIG.3B) E. coli labeled externally with goat anti-E. coli O157:H7 primaryantibody and rabbit anti-goat CY2 labeled secondary antibody; FIG. 3C)Merged A and B images; FIG. 3D) S. aureus labeled internally with EUB338CY3 16S rRNA FISH probe; FIG. 3E) S. aureus labeled externally withbiotin conjugated rabbit anti-S. aureus primary antibody andstreptavidin conjugated fluorescein isothiocyanate (FITC) secondaryantibody; FIG. 3F) Merged D and E images; Scale bar is 5 μm.

FIGS. 4A-4D. Cytometric bead fluorescence images with or without E. coliO157:H7. FIG. 4A) Standard assay with non-spiked PBS; FIG. 4B) Standardassay with non-spiked spinach rinse; FIG. 4C) Standard assay with E.coli O157:H7; FIG. 4D) CAT-FISH assay with E. coli O157:H7. Negativecontrols (no target and/or no reporter) are not shown due to lack offluorescence. Scale bars are 10 μm.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an oligonucleotide hybridization probe that can be usedaccording to the present invention.

SEQ ID NO:2 is an oligonucleotide hybridization probe that can be usedaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns materials and methods for detecting atarget cell in a population. Methods of the invention compriseinternally labeling cells via FISH in a liquid system using one or moredetectably labeled probes that target rRNA, such as 16S and/or 23S rRNAof prokaryotes, followed by binding of capture antibodies targeted (CAT)for specific cell surface epitopes. In one embodiment, the target cellsare bacterial cells. FISH methods and probes that target 16S rRNA aredescribed in U.S. Pat. No. 7,771,941, the disclosure of which isincorporated by reference herein in its entirety.

The subject invention provides for detection of target cells in complexsamples using the dual specificity provided by rRNA sequences (nucleicacid level specificity) and the epitope specific binding of antibodies(protein level specificity). The FISH methods are modified to allow forretaining samples in liquid form and to permit subsequent antibodybinding to the target cell surface. As the targeted cells are internallylabeled by the FISH probes, their capture by antibodies and subsequentdetection are platform independent as long as the detector is capable ofdetecting the label, e.g., fluorescent signal in the appropriatewavelength. The detection methods allow for capture of FISH-labeledtarget cells onto antibody-coupled beads and subsequent detection usingcytometric identification, in a multiplex capable format.

In one embodiment of a method of the invention, a sample to be testedfor the presence of target cells therein is obtained. Optionally, cellsin the sample can be centrifuged or otherwise isolated from the sample.The cells are then fixed in a solution that will allow for antibodybinding after the FISH processing steps (e.g., a non-crosslinkingfixative). In a specific embodiment, the cells are fixed with amethacarn solution. In one embodiment, following fixation, the cells canoptionally be contacted with one or more lytic enzymes, such aslysozyme, lysostaphin, and/or nisin. After fixation, the cells aredehydrated optionally using ethanol washes and air drying. The cells arethen contacted with a detectably labeled probe that targets an rRNA ofthe target cell under suitable hybridization conditions (FISH) and foran effective period of time. In one embodiment, the detectably labeledprobe is an oligonucleotide. In another embodiment, the detectablylabeled probe is a peptide nucleic acid probe. In one embodiment, theprobe targets a 16S and/or 23S rRNA of a prokaryote. In a specificembodiment, the probe can be fluorescently labeled. Optionally, thecells are centrifuged after FISH and resuspended in a suitable buffersolution. The cells are then contacted with an antibody immobilized on asurface platform, wherein the antibody (or antibodies) is directed toone or more antigens on the surface of the target cell. The target cellsbind to the antibodies immobilized on the surface platform. The boundtarget cells can be washed with a suitable medium or buffer. The boundtarget cells are then detected via detection of the labeled probehybridized to rRNA of the target cells using any suitable detectionmeans.

Antibodies of the invention can be immobilized on any suitable surfaceplatform. Examples of surface platforms contemplated for use with thesubject invention include, but are not limited to, beads (such asmicrobeads); microtiter plates; microarrays; fiber optic waveguides;planar array biosensors; and microfluidic chips. In an exemplifiedembodiment, the antibody is immobilized on a bead surface, such as amicrobead. In a specific embodiment, the beads are composed of orcomprise a material that can be attracted by a magnetic field. In oneembodiment, the beads are magnetic or superparamagnetic beads. Beadsuseful in the present invention can be color coded using differentratios of different detectable labels or fluorescent molecules, e.g.,orange fluorescent molecules. The detectable label or fluorophore of thebead can be different than the detectable label of the labeled probe.The target cells are bound to the beads via the antibody binding to thetarget cell antigens and then the beads are isolated or separated outfrom unbound material. In those embodiments where the beads can beattracted by a magnetic field, bound target cells can be separated fromthe unbound material by application of a magnetic field which collectsand isolates the beads from the unbound material. In another embodiment,the beads can be sorted from materials using fluorescence activated cellsorting. The labeled probe bound to the target cell rRNA is thendetected using suitable detection means. Optionally, the beads havingtarget cells bound thereto can be washed and/or resuspended in asuitable medium or buffer prior to application of the detection means.

In one embodiment, a fixative solution useful in the subject inventionis a methacarn solution. Methacarn solution has been found to be anon-cross-linking protein-precipitating fixative that does not appear toaffect polynucleotide or protein analysis of fixed tissues and usuallywill give superior immunohistochemical results (Dotti et al. (2010);Shibutani and Uneyama (2002); Urieli-Shoval et al. (1992)). Otherfixative solutions contemplated within the scope of the inventioninclude, but are not limited to, ethanol, methanol, Carnoy's solution(e.g., 60% ethanol, 30% chloroform and 10% glacial acetic acid), andHistoChoice® MB (AMRESCO, Inc., Solon, Ohio) (U.S. Pat. Nos. 5,429,797and 5,439,667).

Oligonucleotide probes useful in the subject invention can be readilyidentified and prepared by an ordinarily skilled artisan.Oligonucleotide probes that bind to rRNA of a target cell have beenidentified and described in the art. In addition, an ordinarily skilledartisan can identify rRNA sequences of a target cell and can prepareoligonucleotide probes that will hybridize with specificity to thosesequences. Databases for rRNA sequences and probes are available to thepublic (see, for example, Loy et al. (2007) andhttp://www.microbial-ecology.net/probebase; Ribosomal Database Projecthttp://rdp.cme.msu.edu). In one embodiment, an oligonucleotide probe ofthe invention hybridizes with 16S and/or 23S rRNA of a prokaryote. Inanother embodiment, an oligonucleotide probe of the invention hybridizeswith 18S and/or 28S rRNA of a eukaryote. Oligonucleotide probes of theinvention can comprise one or more detectable labels. In one embodiment,an oligonucleotide probe comprises a dual label.

Detectable labels that can be used with a probe of the present inventioninclude, but are not limited to, fluorescent moieties, chemiluminescentand bioluminescent reagents, enzymes, and radioisotopes. Fluorescentmoieties include, but are not limited to, fluorescein, fluoresceinisothiocyanate, Cascade Blue, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride, Texas Red, Oregon Green, cyanines (e.g.,CY2, CY3, and CY5), umbelliferone, allophycocyanine or phycoerythrin. Anexample of a luminescent material includes luminol. Examples ofbioluminescent materials include, but are not limited to, luciferin,green fluorescent protein (GFP), enhanced GFP (Yang et al., 1996), andaequorin. Enzymes that can be used include but are not limited toluciferase, beta-galactosidase, acetylcholinesterase, horseradishperoxidase, glucose-6-phosphate dehydrogenase, and alkaline phosphatase.If the detectable label is an enzyme, then a suitable substrate that canbe acted upon by the enzyme can be used for detection and measurement ofenzyme activity. In one embodiment, if the detectable label is aperoxidase, the substrate can be hydrogen peroxide (H₂O₂) and 3-3′diaminobenzidine or 4-chloro-1-naphthol and the like. Other substratessuitable for use with other enzymes are well known in the art. Isotopesthat can be used include, but are not limited to, ¹²⁵I, ¹⁴C, ³⁵S, and³H.

Antibodies that can be used in the subject invention can be genus orspecies specific to a target cell. Antibodies of the invention can beprepared using standard techniques known in the art. Antibodies usefulin the invention can be polyclonal or monoclonal antibodies. Monoclonalantibodies can be prepared using standard methods known in the art(Kohler et al., 1975).

An antibody that is contemplated for use in the present invention can bein any of a variety of forms, including a whole immunoglobulin, anantibody fragment such as Fv, Fab, and similar fragments, as well as asingle chain antibody that includes the variable domain complementaritydetermining regions (CDR), and similar forms, all of which fall underthe broad term “antibody,” as used herein.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment of an antibody yields an F(ab′)₂fragment that has two antigen binding fragments, which are capable ofcross-linking antigen, and a residual other fragment (which is termedpFc′). Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “antigen binding fragment” withrespect to antibodies, refers to, for example, Fv, F(ab) and F(ab′)₂fragments.

Antibody fragments can retain an ability to selectively bind with theantigen or analyte are contemplated within the scope of the inventionand include:

(1) Fab is the fragment of an antibody that contains a monovalentantigen-binding fragment of an antibody molecule. A Fab fragment can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain.

(2) Fab′ is the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain. Two Fab′ fragmentsare obtained per antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region.

(3) (Fab′)₂ is the fragment of an antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction. F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain (V_(L)), thevariable region of the heavy chain (V_(H)), linked by a suitablepolypeptide linker as a genetically fused single chain molecule. Suchsingle chain antibodies are also referred to as “single-chain Fv” or“sFv” antibody fragments. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv fragments, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y.,pp. 269 315 (1994).

Antibodies within the scope of the invention can be of any isotype,including IgG, IgA, IgE, IgD, and IgM. IgG isotype antibodies can befurther subdivided into IgG1, IgG2, IgG3, and IgG4 subtypes. IgAantibodies can be further subdivided into IgA1 and IgA2 subtypes.

Examples of bacterial cells that can be detected using the presentinvention include, but are not limited to, Nitrospira spp., Nitrosospiraspp., Nitrobacter spp., Nitrosomonas spp., Clostridium spp., Bacillusspp. (such as Bacillus anthracis), methanogenic archaea, coliforms (suchas E. coli), Salmonella spp., Bacteroides spp., Staphylococcus spp.,Streptococcus spp., Neisseria spp., Haemophilus spp., Bordetella spp.,Listeria spp., Mycobacterium spp., Shigella spp., Pseudomonas spp.,Brucella spp., Treponema spp., Mycoplasma spp., Yersinia spp.,Vibrionaceae spp., Chlamydia spp., Legionella spp., Escherichia spp.,Acinetobacter spp., Burkholderia spp., Thiobacillus spp., Rickettsiaspp., Sphinomonas spp., Francisella spp., Campylobacter spp., andHelicobacter spp.

In one embodiment, an oligonucleotide probe of the invention may be onethat specifically hybridizes with at least 8, 9, 10, 11, 12, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides of the targetsequence (such as the 5′ or 3′ end of precursor 16S rRNA; or theinterior region of both precursor 16S rRNA and mature 16S rRNA). Variousdegrees of stringency of hybridization can be employed. The morestringent the conditions, the greater the complementarity that isrequired for duplex formation. Stringency can be controlled bytemperature, salt concentration, chaotropic agent concentration and thelike. Preferably, hybridization is conducted under low, intermediate, orhigh stringency conditions by techniques well known in the art, asdescribed, for example, in Keller and Manak (1987) or Maniatis et al.(1982).

In general, hybridization and subsequent washes can be carried out underintermediate to high stringency conditions that allow for detection oftarget sequences with homology to the exemplified polynucleotidesequence. For double-stranded DNA gene probes, hybridization can becarried out overnight at 20-25° C. below the melting temperature (T_(m))of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/mldenatured DNA. The melting temperature is described by the followingformula (Beltz et al. (1983)).

Tm=81.5° C.+16.6 Log(Na⁺)+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash);

(2) once at T_(m)−20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS(intermediate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20° C. below the melting temperature (T_(m)) of the hybrid in6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. T_(m)for oligonucleotide probes can be determined by the following formula:

T_(m)(° C.)=2(number T/A base pairs)⁺4(number G/C base pairs)(Suggs etal. (1981)).

Washes can be carried out as follows:

(1) twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (lowstringency wash);

(2) once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%SDS (intermediate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment >70 or so bases in length, the followingconditions can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or 2×SSPE, 42° C.

Intermediate: 0.2× or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

Samples contemplated within the scope of the invention include, but arenot limited to, environmental (e.g., water, soil, slurry, sludge, gas,liquid, vapor, etc.) and biological samples. Biological samples can beobtained from an individual. The biological sample may be obtained byany method known in the art. Samples may be collected at a single timepoint or at multiple time points from one or more tissues or bodilyfluids. The tissue or fluid may be collected using standard techniquesin the art, such as, for example, tissue biopsy, blood draw, orcollection of secretia or excretion from the body. Examples of suitablebodily fluids or tissues from which an infectious agent, or componentthereof, may be isolated include urine, blood, intestinal fluid, edemafluid, saliva, lacrimal fluid (tears), inflammatory exudate, synovialfluid, abscess, empyema or other infected fluid, cerebrospinal fluid,pleural effusions, sweat, pulmonary secretions, seminal fluid, feces,bile, intestinal secretions, or any infected tissue including, but notlimited to liver, intestinal epithelium, spleen, lung, pericardium,pleura, skin, muscle, synovium, cartilage, bone, bone marrow, thyroidgland, pancreas, brain, prostate, ovaries, endometrium, uterus, uterinecervix, testes, epididymis, bladder wall, kidney, adrenal, pituitarygland, adipose cells/tissue, omentum, or other cells and tissue. Thefrequency of obtaining one or more biological samples can vary.

A surface platform can have a fixed organizational support matrix thatpreferably functions as an organization matrix, such as a microtitertray. Solid support materials include, but are not limited to, glass,polacryloylmorpholide, silica, controlled pore glass (CPG), polystyrene,polystyrene/latex, polyethylene, polyamide, carboxyl modified teflon,nylon and nitrocellulose and metals and alloys such as gold, platinumand palladium. The solid support can be biological, non-biological,organic, inorganic, or a combination of any of these, existing as beads,particles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides, etc.,depending upon the particular application. Other suitable solidsubstrate materials will be readily apparent to those of skill in theart. The surface of the solid substrate may contain reactive groups,such as carboxyl, amino, hydroxyl, thiol, or the like for the attachmentof nucleic acids, proteins, etc. Surfaces on the solid substrate willsometimes, though not always, be composed of the same material as thesubstrate. Thus, the surface can be composed of any of a wide variety ofmaterials, for example, polymers, plastics, resins, polysaccharides,silica or silica-based materials, carbon, metals, inorganic glasses,membranes, or any of the above-listed substrate materials.

The subject invention also concerns kits comprising in one or morecontainers: a probe that can bind to or hybridize with an rRNA of atarget cell, and optionally a surface platform that can have an antibodyattached thereto. In one embodiment, a kit comprises a detectablylabeled oligonucleotide probe that can hybridize with the target cellrRNA. In one embodiment, a probe of the kit binds to or hybridizes witha 16S and/or 23S rRNA of a target cell. In one embodiment, the kitfurther comprises an antibody, or an antigen binding fragment thereof,that can bind to one or more surface antigens on the surface of thetarget cell. In one embodiment, the antibody is a monoclonal antibody,or an antigen binding fragment thereof. In another embodiment, theantibody is a polyclonal antibody, or an antigen binding fragmentthereof. In one embodiment, the kit further comprises a fixativesolution for fixing cells, wherein the fixative solution allows forantibody binding to one or more antigens on the surface of the cells. Ina further embodiment, the fixative solution is a non-cross linkingfixative. In a specific embodiment, the fixative solution is a methacarnsolution. In one embodiment, the surface platform provided in the kit isa bead, such as a microbead. In a further embodiment, the bead can beattracted by a magnetic field. In a further embodiment, the bead is amagnetic or super paramagnetic bead. In one embodiment, the beadcontains a detectable label or fluorophore. In one embodiment, the probeis labeled with a fluorescent label, a chemiluminescent reagent, abioluminescent reagent, an enzyme, or a radioisotope. In one embodiment,the kit further comprises one or more lytic enzymes. Kits of theinvention can optionally include physiologically acceptable carriersand/or diluents. In one embodiment, a kit of the invention includesinstructions or packaging materials that describe how to use thecomponents of the kit for detection of a target cell. Containers of thekit can be of any suitable material, e.g., glass, plastic, metal, etc.,and of any suitable size, shape, or configuration.

Materials and Methods Microorganisms

Escherichia coli O157:H7 (ATCC 35150) and Staphylococcus aureus (ATCC25923) were cultured at 37° C. with shaking in brain heart infusionbroth (BHI) (Difco, Becton Dickinson, Franklin Lakes, N.J.). Cells werecultured for ˜18 h prior to experiments and then diluted 1:100 in freshBHI and incubated as described. Cells were harvested at 0.8 O.D. (600nm) and washed once with 1× phosphate buffered saline (PBS −11.9 mMphosphate buffer, 137 mM sodium chloride, 2.7 mM potassium chloride, pH7.4 (Fisher BioReagent, Suwanee, Ga.)). Cell pellets were then fixedimmediately (PBS samples) or diluted into complex matrix prior tofixation.

Fixation

Harvested bacteria were fixed by one of two methods: Control cells werefixed with 4% ice-cold paraformaldehyde (PFA) for 2 hrs at 4° C.,collected by centrifugation (9,000×g) for 90 s, decanted and resuspendedin 50% (v/v) ethanol in 1×PBS according to standard FISH protocols(Daims et al., 2005). Test cells were fixed with methacarn solution(6:3:1 parts absolute methanol, chloroform, and glacial acetic acid)made fresh daily (Leong, 1994). All manipulations involving themethacarn solution were performed in a chemical hood. Cell pellets wereresuspended in methacarn, incubated at room temperature (22° C.) for 10min, collected by centrifugation (9,000×g) for 90 s, decanted, andwashed once in 80% (v/v) ethanol and once in 100% ethanol. Cell pelletswere dried in the dark via rotation for 20 min at 46° C. to drive offremaining ethanol (Enviro-Genie, Scientific Industries, Inc., Bohemia,N.Y.). Samples containing spinach rinse (spiked with E. coli O157:H7 orun-amended) were processed in the same manner. For Staphylococcussamples, prior to alcohol dehydration steps, cells were treated withlytic agents as follows: after centrifugation, cells were washed once in10 mM Tris HCl pH 8.0, centrifuged and resuspended in lysozyme (1 mg/mlin 10 mM Tris HCl pH. 8.0) and incubated 15 min at 30° C.; then cellswere collected as described and resuspended in either lysostaphin (10μg/ml in 10 mM Tris HCl pH 8.0) and incubated 5 min at 30° C. or nisin(0.1 g of 2.5% powder in 10 ml 0.02N HCl pH 2.0) diluted into PBS forfinal concentration of 25 μg/ml).

Liquid Fluorescence In Situ Hybridization (LQ-FISH)

Fluorescently-labeled oligonucleotide hybridization probes targeting the16S rRNA for the domain Eubacteria (EUB338; 5′ gctgcctcccgtaggagt 3′ SEQID NO:1) and species S. aureus (SAU349; 5′ gaagcaagcttctcgtccg 3′ SEQ IDNO:2) were obtained from Molecular Probes (Invitrogen, Carlsbad, Calif.)and labeled with either Alexa Fluor 532 or Cy3 at the 5′ end (Loy etal., 2007). Alexa Fluor 532 labeled probe was used for cytometric beadarray assays, and Cy3 labeled probe was used for optimization assays,image capture and image analysis. The probes were diluted to 50 ng/μlwith dH₂O, and stored in 100-μl aliquots at −20° C. in the dark. Fixedand dried sample pellets (pure cultures or spiked matrix) wereresuspended in 200 μl of hybridization buffer containing the labeledprobe at a final concentration of 5 ng/μl. The hybridization buffer alsocontained 20% (v/v) formamide, 0.9 M NaCl, 0.1% SDS and 100 mM Tris HCl(pH 7.0) (Daims et al., 2005). Hybridization was conducted in the darkfor 1 h in a 46° C. water bath. Subsequently, samples were centrifuged(9,000×g) for 90 s, the supernatant was removed, and cells wereresuspended in 500 μl of pre-warmed washing buffer followed by 10 minincubation in a 48° C. water bath in the dark. The washing buffercontained 215 mM NaCl, 20 mM Tris HCl (pH 7.0) and 5 mM EDTA (Daims etal., 2005). After washing, samples were centrifuged and the supernatantremoved. Treated pellets were resuspended in PBS for downstreamprocessing (microscopy, antibody labeling, or immunomagnetic capture andcytometric analysis).

Standard Fluorescence In Situ Hybridization

Variations in oligonucleotide conferred fluorescence intensity basedupon fixative affect were measured. Cells fixed by either PFA ormethacarn were placed on 10-well heavy Teflon coated microscope slides(Cel-Line Associates, New Field, N.J.) and processed by standard FISHprotocols (Amann et al., 1990; Oerther et al., 2000) using theCy3-labeled EUB338 probe. Lytic agents were applied to theStaphylococcus samples as described in the section entitled “Fixation.”The hybridization step was 1 h and the washing step was 30 min. Cellswere counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at aconcentration of 1 μg/mL for 1 minute, rinsed with dH₂O, air dried, andmounted with Cargille immersion oil (Type FF, Cedar Grove, N.J.) and acover slip.

Antibody Binding Assays

Fixed cells were used in adsorption enzyme linked immunosorbent assays(ELISA) to compare antibody recognition after the different fixationtechniques. Bacteria were fixed with either PFA or methacarn asdescribed above and split, with half used as controls and half processedalike through the entire liquid FISH procedure. Additional controlsconsisted of untreated cells that were harvested and stored on ice inPBS from the same initial test cultures. After treatments, cells wereenumerated by direct microscopic count, normalized to 1×10⁸ cells/ml andserially diluted to 10³ cells/ml in PBS. Preliminary experimentsindicated that cell concentrations below 10² cells/ml did not producemeasurable signal and were omitted from further tests (data not shown).Cells were then applied in triplicate to an ELISA plate, incubated at 4°C.˜18 hrs and processed using a QuantaBlu Fluorogenic Peroxidase kit(PIERCE, Rockford, Ill.) according to manufacturer's instructions withthe following exceptions: volumes for each step were normalized to 100μl and incubation times were reduced to 30 min each. Reporter antibodiesincluded affinity purified peroxidase labeled polyclonal goat anti-E.coli O157:H7 (1 μg/ml) (KPL, Gaithersburg, Md.), purified rabbit anti-S.aureus (10 μg/ml) (AbD Serotec, Raleigh, N.C.) and affinity purifiedperoxidase labeled goat anti-rabbit (1 μg/ml) (KPL). ELISA plates werewashed using an EL_(x)50 auto-strip washer (Bio-Tek Instruments Inc.,Winooski, Vt.) and end product detection performed on a SpectraMaxGeminiXS Fluorometer (Molecular Devices, Silicon Valley, Calif.).Results are reported as signal to noise (S/N) ratios for test samplesversus PBS, where an S/N ratio above 3 indicates a positive detectionresult.

Simultaneous FISH and Reporter Antibody Labeling

E. coli O157:H7 and S. aureus samples were individually fixed inmethacarn and processed according to the liquid FISH method described inthe section entitled “Liquid fluorescence in situ hybridization(LQ-FISH)” either with or without Cy3-labeled EUB338 probe. Treatedcells were resuspended in 500 μl PBS containing either 4 μg/ml goatanti-E. coli O157:H7 (KPL—for E. coli O157:H7 samples) or 4 μg/ml biotinconjugated rabbit anti-S. aureus (AbD Serotec—for S. aureus samples),and incubated at room temperature for 30 min in the dark with end overend rotation (24 rpm). The cells were recovered by centrifugation(9,000×g for 90 s) and washed once in 500 μl PBS with 5 min end over endrotation. E. coli and S. aureus samples were then resuspended in 500 μlPBS containing 2 μg/ml Cy2-labeled donkey anti-goat (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) or 2 μg/mlstreptavidin conjugated fluorescein isothiocyanate (FITC) (JacksonImmunoResearch), respectively. Cells were incubated at room temperaturefor 30 min in the dark with end over end rotation, washed, resuspendedin 100 μl PBS, and visualized using an epifluorescent microscope.

Cytometric Assays

A method was developed to incorporate immunomagnetic separation (IMS)with cytometric bead arrays for direct detection of FISH labeled cells(IMS-CAT-FISH) using a specialized flow cytometer. This method is amodification to standard cytometric bead array (CBA) assays, which relyon a labeled detector antibody for target detection. MagPlexC magneticmicrobeads (6.5±0.2 μm, fluorescence region 33, Luminex, Austin, Tex.)were used for all assays, and for all manipulations beads were separatedfrom liquid solutions using the 3-in-1 Magnetic Particle Separator(PureBiotech, LLC, Middlesex, N.J.). Magnetic microspheres were coupledto goat anti-E. coli O157:H7 antibody (KPL) or rabbit anti-S. aureus(AbD Serotec) using the amine coupling kit (BioRad, Hercules, Calif.),per the manufacturer's instructions and stored at 4° C. until used.

Coupled magnetic microspheres were used to capture target cells fromsample suspensions following the LQ-FISH procedure. Antibody-coupledmicrobeads (1,000 per sample) were added to 1 ml of sample and brieflymixed by vortex action on low setting. Mixed samples were centrifuged(6,000×g, 60 s) and target allowed to bind during a 5 min stationaryincubation at 22° C. Samples were briefly mixed after incubation andbeads were separated from the supernatant. Recovered beads were washedtwice with 500 μl PBS containing 0.05% Tween 20 (PBST) in the dark using5 min rotations. After washing steps, beads were separated from thesample supernatant and resuspended in 100 μl PBST. Beads were placedinto 96-well round-bottom plates, and analyzed using the Bio-Plex 200reader (BioRad), per the manufacturer's instructions using the high PMTsetting. Fluorescence signals were expressed as the mean fluorescenceintensity of 100 beads per well.

Positive signals were determined using signal above limit of detection(SALOD) values. SALODs were calculated by taking the average samplesignal and subtracting the limit of detection (LOD) value. The LOD valuewas calculated by averaging sample blanks and adding three times thestandard deviation of the sample blanks. All experiments were performedin duplicate or triplicate.

Simulation of Complex Testing Situations Spinach Rinse MatrixPreparation

Spinach rinse was generated to simulate a field wash sample as E. coliO157:H7 contamination is a concern in commercial produce products. Freshbundled spinach was purchased from a local wholesale market (Tampa,Fla.) and processed within one day. Spinach rinse was generated andstored at −20° C. until used. Rinses were tested for background E. coliO157:H7 contamination according to established FDA-BAM procedures (Feng,1998), and determined to be devoid of endogenous E. coli O157:H7 priorto initiation of experiments. Serially diluted samples were plated onBHI agar using the spread plate method to determine microbial backgroundloads.

Prior to IMS-CAT-FISH, E. coli O157:H7 was diluted into spinach rinse,mixed 1:1 with 2× modified buffered peptone water with pyruvate (mBPWp),(Feng, 1998), and enriched overnight at 42° C., statically. Afterenrichment, spiked and un-spiked samples were plated onto CT-SMAC agarto verify growth in spiked samples and absence of target in controls.Before sampling, enriched samples were mixed briefly and allowed tostand for 1 min to allow for sand and large particulates to settle out.Samples for analysis were collected from the resultant supernatant, andprocessed for IMS-CAT-FISH, as described.

Blood Cultures

Citrated sheep blood (Fisher Scientific) was used as a surrogate forhuman blood. Blood samples were spiked with S. aureus at concentrationsof ˜2 CFU/ml (verified by plate counts on BHI agar), and diluted 1:5into BACTEC-derived blood culture media, which was prepared in-house andcontained 3% soybean-casein digest, 0.3% yeast extract, 0.01% beefextract, 0.1% sucrose, 0.0005% hemin, 0.00005% menadione, 0.001%Pyridoxal HCL, 0.04% sodium bicarbonate, and 0.035% sodiumpolyanetholsulfonate (all w/v). Samples were enriched overnight at 37°C., with aeration, and plated on BHI agar to verify bacterial growth inspiked samples, and absence of bacteria in non-spiked samples. 500 ul ofthe blood culture sample was used for each assay. The sample was mixedwith 500 ul water and incubated for 5 minutes at room temperature topermit lysis of remaining erythroid cells. Samples were then centrifugedto pellet bacteria, re-suspended in 1 ml PBS, and processed according tothe IMS-CAT-FISH procedure. Prior to IMS capture, samples were diluted1:100 in order to dilute particulates resulting from remaining bloodcomponents.

Microscopic Imaging and Data Analysis

Samples for image analysis were placed on 10-well microscope slides(Cel-Line) and either observed under wet mount or air-dried. Air driedcells were mounted with Cargille immersion oil (Type FF) and a coverslip. Oligonucleotide and/or antibody conferred fluorescence werevisualized with upright epifluorescence microscopes, either a LeitzDiaPlan (Heerbrugg, Switzerland) or an Olympus BX60F (Center Valley,Pa.). Digital images were captured using Spot-FLEX CCD cameras and imagemodifications (for publication purposes only) were performed using SpotSoftware 4.6 (Diagnostic Instruments, Inc., Sterling Heights, Mich.).Image modifications consisted of cropping, scale bar calibrations andpixel multiplicative brightness adjustments. Brightness adjustments werescaled up a factor of 2 or 3 dependant upon image series, however withineach series the multiplicative factor was the same, so that relativeintensities were comparable. All data analysis was performed on rawimages without modifications. Fluorescent images were evaluated with thedaime 1.3.1 software package (Daims et al., 2006). Measure objectsanalysis calculated the number of cells detected, as well as the meanfluorescent intensity and standard deviations for each cell. SIGMAPLOT10 (Systat Software, Inc., San Jose, Calif.) was used for graphicanalysis. Student's t test was used to determine if significantdifferences occurred between fixative types.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Optimization of Processing Conditions

Preliminary evaluations of multiple fixatives and conditions, includingstandard FISH fixation with PFA, did not facilitate sufficientantigen-antibody binding as required for target capture and detection onbiosensors (data not shown). Experiments attempting to ascertainantigen-antibody binding capabilities for stored PFA fixed E. coli cellswere unsuccessful in producing any antibody binding (data not shown).Thus all subsequent experiments used “fresh” fixed cells for evaluationpurposes, in that cells were processed immediately after fixation stepswithout holding or storage. Methacarn fixation was the most promisingcandidate for both fluorescence and antibody binding and wassubsequently optimized for use in the LQ FISH protocol.

Example 2—Influence of Fixative Upon Fluorescent Signal

E. coli O157:H7 and S. aureus were fixed in either methacarn or PFA todetermine if methacarn fixation produced cells capable of binding probeand emitting fluorescence signals comparable to standard protocols.Cells from both fixative treatments (methacarn and PFA) were mounted onslides and subjected to standard FISH. In addition, cells were processedby the LQ-FISH method using methacarn fixation, and spotted ontoseparate slides (FIG. 1). Relative fluorescence intensity means for E.coli O157:H7 cells (200 per treatment) were: 61±12 for PFA, 68±11 formethacarn and 58±10 for LQ-FISH. Analyses indicated that there were nosignificant differences between the PFA and LQ-FISH treatments, but themethacarn treated cells were significantly brighter (p<0.0004) than bothPFA and LQ-FISH cells. Relative fluorescence intensity means for S.aureus cells (150 per treatment) were: 48±14 for PFA, 56±18 formethacarn and 100±28 for LQ-FISH. Analyses indicated that the PFAtreated cells were significantly dimmer (p<0.003) than both methacarnand LQ-FISH cells, and LQ-FISH treated cells were significantly brighterthan the slide based methacarn treated cells (p<0.003).

Example 3—Influence of Fixative Upon Antibody Binding

Adsorbent ELISAs were used to evaluate PFA and methacarn fixationeffects. In addition, fixed cells were subjected to furtherhybridization conditions (LQ FISH) to determine the effect ofhybridization treatments on antibody binding for E. coli O157:H7 and S.aureus (FIG. 2). Antibody binding was significantly reduced for PFAfixed E. coli as compared to control (non-fixed) and methacarn fixedcells for all concentrations below 10⁸ cells/ml (FIG. 2A). Positive S/Nratios were observed for methacarn, control, and PFA at 10⁴, 10⁵, and10⁶ cells/ml, respectively. Methacarn treatment achieved better bindingthan controls at concentrations below 10⁵ cells/ml and showed nodifference at higher concentrations. After LQ-FISH hybridizationtreatments, methacarn treated cells produced higher S/N ratios than PFAtreated cells; however, the minimum concentration producing a positivesignal was one log higher as compared to non-hybridized cells (FIG. 2B).No change in minimum concentration was observed for the PFA treatedcells after hybridization. Antibody binding for S. aureus produced nosignificant differences between controls and methacarn or PFA fixedcells, and positive S/N ratios were achieved at 10⁶ cells/ml for allconditions (FIG. 2C). After hybridization, the methacarn fixed cellsproduced slightly higher S/N ratios, but were not significantlydifferent from PFA samples. (FIG. 2D).

Example 4—Simultaneous Visualization of Oligonucleotide and AntibodyFluorescence

Both labeling methods were used in conjunction to examine labelingcoverage after methacarn fixation with LQ-FISH hybridization. E. coliO157:H7 and S. aureus were simultaneously labeled with rRNA probes andantibodies and generated signals that were sufficient for microscopicvisualization (FIG. 3). Appropriate un-labeled controls were included(±probe and/or antibody) and verified (data not shown). Visualexamination of E. coli cells indicated that they were uniformly labeledwith both CY3 FISH probe and CY2 antibodies, providing ample coveragefor dual level specificity detection. S. aureus cells also exhibiteddual labeling, but the signal intensities appeared to be more variablefor both fluorescent markers.

Example 5—Immunomagnetic Cytometric Bead Array Detection

A cytometric bead array biosensor was selected as an example platform todemonstrate the utility of the dual specificity target detection. Cellswere labeled using LQ-FISH and captured by IMS using antibody-coupledmagnetic cytometric array microspheres (IMS-CAT-FISH). Bead-associatedfluorescence signal was then determined using the cytometric arrayreader. Initially, limit of detection assays for E. coli O157:H7 and S.aureus were performed in PBS, and microscopic evaluation of E. coliO157:H7 was used to visually verify labeling and capture. Utility of themethod was subsequently verified using a more complicated testing matrixappropriate for each organism. In addition, S. aureus limit of detectionassays were performed using either species specific lytic treatments(lysostaphin) or general gram-positive lytic treatment (nisin),incorporated into the LQ-FISH procedure.

Example 6—E. Coli O157:H7 Detection Assays

We previously reported that standard cytometric bead array (IMS-CBA)assays using labeled detector antibodies are useful for rapid detectionof E. coli O157:H7 in both PBS and a more complex testing scenario (rawspinach testing via enrichment of rinse samples) (Leach et al., 2010).However, in spinach rinse enrichment samples, some non-specificbackground signals were encountered, suggesting the need for a morespecific detection procedure (Leach et al., 2010). Toward this end,CAT-FISH was applied.

Microscopic evaluation was first conducted to evaluate target captureand labeling for both the standard IMS-CBA assay (antibody label) andthe IMS-CAT-FISH method (FISH label). All samples (bead only controls,un-spiked PBS blanks, spiked PBS samples, and un-spiked spinach rinseblanks) were spotted onto slides and examined (FIG. 4). Beads incubatedwithout target or reporter produced no detectable signal in either assay(not shown). Detector-associated signal for PBS blanks was absent fromCAT-FISH treatments (i.e., no labeled cells present, not shown), but adim signal was visible after processing according to standard arrayassay method (FIG. 4A). Beads incubated with control cells producedhighly visible signal for both treatments. E. coli detected with thestandard assay produced images with visible halos surrounding thesurface of the captured cells, corresponding to the binding of labeleddetector antibody to bead-capture cells. Labeled cells were easilydistinguishable from the bead surfaces (FIG. 4C). E. coli cells detectedwith CAT-FISH produced images of solidly labeled cells bound to thesurface of the beads (FIG. 4D). Non-spiked spinach blanks treated withCAT-FISH produced no detectable signal (not shown). Interestingly, therewas observable signal on beads after the standard assay was performed onthe un-spiked rinse. Fluorescence images displayed relatively largepatches of labeled non-target particulate material attached to thecytometric array beads (FIG. 4B), thus demonstrating non-specificbinding of spinach debris to the beads and the reporter antibody.

As microscopic evaluation indicated successful labeling and capture, acytometric array biosensor (BioPlex 200) was used to evaluate targetsignals obtained using the IMS-CAT-FISH assay. Detection wassporadically observed at 10³ cells/ml and routinely observed at 10⁴cells/ml (Table 1). A one log increase in signal strength was observedfor each log increase in cell concentration until saturation was reachedat 10⁶ cells/ml, after which no significant increases were observed.These detection limits are similar to those previously reported for thestandard cytometric array assay (Kim et al., 2009; Kim et al., 2010;Leach et al., 2010) using a reporter antibody.

Next, the ability of the CAT-FISH dual labeling technique to mitigatematrix and antibody cross-reactivity problems was tested using enrichedspinach rinse cultures. Two independent batches of spinach rinse wereprepared according to the standard FDA-BAM protocol (Feng, 1998), andeither left un-spiked or spiked with 0.9 CFU/ml E. coli O157:H7. Afterovernight enrichment, all samples were processed according to theCAT-FISH method, and FISH-labeled target cells were captured usinganti-E. coli O157:H7 coupled magnetic cytometric array beads. Averagerelative fluorescent signals reported were 21.4+/−2.3 for PBS controls(n=3), 26.1+/−6.2 for un-spiked samples (n=12), and 583.1+/−200.6 (n=12)for spiked samples.

Example 7—S. Aureus Detection Assay

S. aureus samples were treated with either a species specific or ageneralized lytic agent to improve probe accessibility. Both weredemonstrated as potential modifications of the assay, dependent uponwhether the presence of Staphylococcus species is suspected (e.g., inblood samples gram positive cocci growing in clusters are assumed to beStaphylococcus species). PBS was spiked with targeted concentrations of5×10⁷ cells/ml S. aureus (direct count) and serially diluted to 10¹cells/ml with PBS. Final spike concentrations were confirmed by BHIplate count at 4.9±1.3×10⁷ CFU/ml.

IMS-CAT-FISH assays produced consistent positive signal for all cellconcentrations greater than 10³ cells/ml for both lysostaphin and nisin(Table 1). Control cells (absent probe) did not produce any positivesignals. A one log increase in signal strength was observed for each logincrease in cell concentration until saturation was reached at 10⁶cells/ml, after which signal decreases were observed. A two-fold highersignal per concentration was generated using the Staphylococcus specificlysostaphin than was observed with the nisin.

As a further test for process utility, S. aureus was spiked into sheepblood at approximately 2 CFU/ml (verified by viable counts) to simulatea low-level bloodstream infection. Samples were cultured overnightaccording to standard blood culture practices and further processed, asdescribed. In preliminary experiments, antibody cross-reactivity fornon-aureus Staphylococcus species was observed (not shown), so thedual-level specificity allowed by CAT-FISH was demonstrated through theuse of an S. aureus-specific probe. Two independent blood culturesamples were each processed in triplicate according to the IMS-CAT-FISHmethod. The average relative fluorescent signals generated after capturewere 35.2+/−2.1 for buffer controls (n=3), 32.4+/−4.3 for un-spikedblood controls (n=6), and 562.2+/−106.4 for S. aureus samples (n=6).

TABLE 1 CAT-FISH Detection of E. coli O157:H7 or S. aureus in PBS usinga cytometric bead array biosensor. S. aureus E. coli O157:H7 LysostaphinNisin Signal SALOD^(a) Signal SALOD Signal SALOD PBS Buffer 13.9 ± 1.217.3^(b) 32.7 ± 2.5  40.3^(b) 32.7 ± 2.5  40.3^(b) 10¹/ml — — 31.9 ±3.5  −8.3 31.2 ± 4.8  −9.1 10²/ml — — 38.2 ± 10.6 −2.0^(c) 37.7 ± 14.6−2.6^(c) 10³/ml 19.3 ± 6.2 2.0^(c) 125.7 ± 80.5  85.4 67.4 ± 33.1 27.110⁴/ml  48.4 ± 18.0 31.0 ^(d) 646.7 ± 196.5 606.4 364.0 ± 180.7 323.710⁵/ml  365.9 ± 179.0 348.6 3370.2 ± 591.6  3329.9 1650.7 ± 670.4 1610.4 10⁶/ml 2277.8 ± 658.7 2260.5 5464.8 ± 1086.3 5424.5 2923.2 ±1178.9 2882.9 10⁷/ml 2163.4 ± 642.0 2146.0 3318.3 ± 1509.2 3278.0 2498.6± 1338.3 2458.3 ^(a)SALOD—Signal Above Limit of Detection^(b)LOD—Baseline Limit of Detection value used to calculate sample SALODvalues ^(c)Sporadically positive ^(d)Bold numbers indicate positiveSALOD values

Example 8

A new method for simultaneous detection and identification of pathogensis presented using a modified fluorescence in situ hybridizationtechnique, which internally labels targeted nucleic acids, while stillpermitting antibody binding to surface antigens. This dual levelspecificity permits two levels of accuracy in determining presence orabsence of pathogens within a complex matrix. Comparison of LQ-FISHprocessing to standard PFA slide based FISH indicated that comparablefluorescent signal was obtained for both E. coli O157:H7 and S. aureuscells. This denotes that the fixation and hybridization processing stepsof CAT-FISH work with both Gram positive and Gram negative bacteria.While the fluorescent intensity of the Staphylococcus cells tended to bemore variable than those of E. coli, this is not unexpected asstaphylococci are known for hybridization difficulties due to membranepermeability issues (Amann et al., 1990; Roller et al., 1994).Permeability issues are typically overcome by use of lytic enzymetreatments prior to hybridization (Roller et al., 1994; Hogardt et al.,2000; Peters et al., 2006), and the use of lytic enzymes was observed toimprove signal intensity for S. aureus in our assays (not shown). WhileKempf et al. demonstrated the utility of lytic treatments; they alsoindicated that permeabilization can lead to cell destruction duringcentrifugation (Kempf et al., 2000; Kempf et al., 2005). However, withcareful modulation, this can be overcome, as demonstrated here, andimproved signal can be generated for gram-positive targets. In thisstudy, both specific and general lytic agents were used to demonstrateimproved probe accessibility options dependent upon whether or not thespecific target is known to the investigator. As a further potentialmodification, Hartmann et al. have demonstrated the utility of peptidenucleic acid probes (PNA FISH) for overcoming staphylococcalpermeability problems.

Methacarn treatment of E. coli allowed better antibody binding than thatobserved for untreated cells and two log better binding than that ofcells treated with PFA prior to hybridization. After hybridizationtreatments, the methacarn treated cells still out performed PFA treatedcells, but a one log reduction in sensitivity was observed (as comparedto non-hybridized methacarn treated cells). This is likely due to theinclusion of formamide in the hybridization solution. The similarity insensitivity for PFA treated cells (pre vs. post hybridization) suggeststhat the surface antigens were already damaged sufficiently, such thatno further reduction in antibody binding was observed after formamideaddition. Although possible to reformulate the hybridization conditionsto maintain stringency with the exclusion of formamide, this woulddeviate from standard practices, and was out of the scope of this study(Stahl and Amann, 1991). The lack of significant differences betweentreatments for the S. aureus cells is likely due to the cell membraneand permeability issues as discussed previously. The two log decrease insensitivity for S. aureus as compared to E. coli is most likely due topoorer quality of the antibody, since a 10-fold more concentrated stockwas necessary to achieve detection, even for the untreated control.

LQ-FISH and antibody binding conditions were evaluated separately, withantibody binding assessed using a standard ELISA kit that was not basedon FISH signal. Once both conditions were optimized and detectionconfirmed individually, both detection types were combined (CAT-FISH)and samples examined for labeling coverage. Both E. coli and S. aureuswere sufficiently labeled by probe and antibody for easy visualdetection. Whereas E. coli cells were fairly uniform in coverage, thereseemed to be an alternating pattern for S. aureus. S. aureus cells thathad high 16S rRNA signal had low antibody signal and vice versa. Thismay be due in part to growth phase of the cells in regard to ribosomalcontent and protein expression or a limitation to the current protocol.

The potential for CAT-FISH utility was further demonstrated on acommercially available cytometric bead array biosensor. CAT-FISH treatedcells were successfully captured by magnetic array beads (IMS-CAT-FISH),and detected via signals emitted from the fluorescent probe. The BioPlex200 was selected due to its popularity and the versatility of cytometricbead arrays, overall, but it should be noted that this platform is notoptimized for detection of the CAT-FISH signal. Specifically, theBioPlex 200 is pre-set and optimized for the detection of cellsexternally labeled using a phycoerythrin-tagged antibody, not internallylabeled with Alexa Fluor 532. The fluorophore used was one that matcheddetector specifics as closely as possible but it was not possible toexactly match the optimum detection conditions, since the excitation andemission wavelengths of the reader are pre-set and could not be altered.

The aforementioned compatibility discrepancies not withstanding, similardetection limits were achieved with IMS-CAT-FISH as compared to thestandard assay for E. coli O157:H7 in PBS buffer, and similar detectionlimits for S. aureus were also observed. This suggests that methacarnfixation permits sufficient target capture from a fluid matrix usingantibody-coupled beads, and LQ-FISH generated fluorescent signals wereample enough to result in minimal to no loss in sensitivity. Notably,preliminary experiments to compare bead binding to PFA treated cellswere unsuccessful in producing any signal and were not pursued.

Inasmuch as there may be problems between how well a detection methodworks in a laboratory buffer sample versus a real-world sample,additional sample matrices were tested. We have previously shown thatdetection of E. coli O157:H7 in enriched spinach rinse samples ispossible using the cytometric array system, albeit with somedifficulties due to background signals likely associated with theantibody used (Leach et al., 2010). We found that CAT-FISH is successfulin detecting E. coli O157:H7 when used in conjugation with establishedspinach testing methods (rinsing and enrichment). It is interesting tonote that microscopic evaluation of samples indicated that non-specificsignals in standard antibody assays are due to binding of debris toantibody-coupled beads, as opposed to non-target cells (FIG. 4B). Thus,in this instance it was possible to use a general probe for the CAT-FISHprocedure (since it labels only cells) to successfully eliminate thebackground problem.

We also evaluated the utility of CAT-FISH for S. aureus detection in anappropriate complex matrix. As bloodstream infections are commonlycaused by S. aureus, we mimicked established blood testing conditions byspiking sheep blood with S. aureus and culturing in standardblood-culture media. We have observed cross-reactivity of our antibodywith other Staphylococcus species (data not shown), so we chose todemonstrate the dual-label utility of CAT-FISH via incorporation of botha specific antibody and a specific probe. Our results indicated thatspecific antibodies can be used in association with specific probes todetect S. aureus in blood cultures via CAT-FISH.

Modification to further improve detection includes mainly optimizationsspecific to individual instruments, such as alignment of probe/detectorexcitation/emission pairs (e.g., BioPlex detection presented herein).Additionally, alteration of types and/or number of oligonucleotideprobes used during hybridization may also be applied, (e.g., duallabeled FISH probe (DOPE-FISH) (Stoecker et al., 2010); peptide nucleicacid probes (Hartmann et al., 2005; Almeida et al., 2010) or multipleprobes targeting different rRNA regions (Amann et al., 1990)). CAT-FISHalso has the potential for transition to kit and/or multi-well plateformats, which would significantly reduce the amount of time requiredfor processing per sample. Furthermore, should additional confirmationbe necessary, treatment of bacterial cells with methacarn does notpreclude assessment by PCR methods, and auxiliary (or alternate)analysis is possible prior to hybridization steps (data not shown).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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We claim:
 1. A method for detecting a target cell in a population ofcells, wherein the method comprises: a) obtaining a sample of cells fromthe population of cells; b) fixing the cells in a solution that willallow for antibody binding to one or more antigens on the surface of thecells after hybridization processing steps; c) dehydrating the fixedcells; d) contacting the fixed cells with a detectably labeledoligonucleotide probe that targets and hybridizes with rRNA of thetarget cell under suitable hybridization conditions; e) contacting thecells after step d) with antibody immobilized on a surface platform,wherein the antibody binds to one or more antigens on the surface of thetarget cell; and f) detecting the detectably labeled oligonucleotideprobe hybridized to rRNA of the target cell.
 2. The method according toclaim 1, wherein cells are isolated from the sample following step a).3. The method according to claim 2, wherein the cells are isolated bycentrifugation of the sample.
 4. The method according to claim 1,wherein the solution in step b) is a non-cross linking fixative.
 5. Themethod according to claim 4, wherein the fixative is a methacarnsolution.
 6. The method according to claim 1, wherein the cells aredehydrated in step c) using one or more ethanol washes and/or airdrying.
 7. The method according to claim 1, wherein the detectablylabeled oligonucleotide probe is labeled with a fluorescent label, achemiluminescent reagent, a bioluminescent reagent, an enzyme, or aradioisotope.
 8. The method according to claim 1, wherein after step d)the cells are centrifuged and resuspended in a suitable buffer one ormore times.
 9. The method according to claim 1, wherein the surfaceplatform is a bead; microtiter plate; microarray; fiber optic waveguide;planar array biosensor; or microfluidic chip.
 10. The method accordingto claim 9, wherein the bead can be attracted by a magnetic field andthe bead having target cells bound thereto is isolated by applying amagnetic field.
 11. The method according to claim 10, wherein the beadis a magnetic or super paramagnetic bead.
 12. The method according toclaim 1, wherein the bound target cells are washed one or more timesprior to detecting the detectably labeled oligonucleotide probe.
 13. Themethod according to claim 1, wherein the antibody is a monoclonalantibody, or an antigen binding fragment thereof.
 14. The methodaccording to claim 1, wherein the antibody is a polyclonal antibody, oran antigen binding fragment thereof.
 15. The method according to claim1, wherein the rRNA is 16S and/or 23S RNA.
 16. The method according toclaim 1, wherein the target cell is a bacterial cell.
 17. The methodaccording to claim 16, wherein the bacterial cell is Nitrospira spp.,Nitrosospira spp., Nitrobacter spp., Nitrosomonas spp., Clostridiumspp., Bacillus spp., methanogenic archaea, coliforms, Salmonella spp.,Bacteroides spp., Staphylococcus spp., Streptococcus spp., Neisseriaspp., Haemophilus spp., Bordetella spp., Listeria spp., Mycobacteriumspp., Shigella spp., Pseudomonas spp., Brucella spp., Treponema spp.,Mycoplasma spp., Yersinia spp., Vibrionaceae spp., Chlamydia spp.,Legionella spp., Escherichia spp., Acinetobacter spp., Burkholderiaspp., Thiobacillus spp., Rickettsia spp., Sphinomonas spp., Francisellaspp., Campylobacter spp., or Helicobacter spp.
 18. The method accordingto claim 1, wherein the hybridization conditions in step d) arestringent hybridization conditions.
 19. The method according to claim 9,wherein the bead is a microbead or wherein the bead contains adetectable label or fluorophore.
 20. A kit for detecting a target cell,wherein the kit comprises in one or more containers: a) a detectablylabeled probe that can bind to or hybridize with rRNA of the targetcell; b) a surface platform that can have an antibody attached thereto.